Unveiling the promise of peptide nucleic acids as functional linkers for an RNA imaging platform.

Linkers in chemical biology provide more than just connectivity between molecules; their intrinsic properties can be harnessed to enhance the stability and functionality of chemical probes. In this study, we explored the incorporation of a peptide nucleic acid (PNA)-based linker into RNA-targeting probes to improve their affinity and specificity. By integrating a PNA linker into a small molecule probe of the Riboglow platform, we enabled dual binding events: cobalamin (Cbl)-RNA structure-based recognition and sequence-specific PNA-RNA interaction.

Cellular zinc status alters chromatin accessibility and binding of p53 to DNA.

Zn is an essential metal required by approximately 850 human transcription factors. How these proteins acquire their essential Zn cofactor and whether they are sensitive to changes in the labile Zn pool in cells remain open questions. Using ATAC-seq to profile regions of accessible chromatin coupled with transcription factor enrichment analysis, we examined how increases and decreases in the labile zinc pool affect chromatin accessibility and transcription factor enrichment.

Protocol for measuring labile cytosolic Zn using an in situ calibration of a genetically encoded FRET sensor.

Zinc (Zn) plays roles in structure, catalysis, and signaling. The majority of cellular Zn is bound by proteins, but a fraction of total Zn exists in a labile form. Here, we present a protocol for measuring labile cytosolic Zn using an in situ calibration of a genetically encoded Förster resonance energy transfer (FRET) sensor.

Protocol for measuring cell cycle Zn dynamics using a FRET-based biosensor.

The exchangeable Zn pool in cells is not static but responds to perturbations as well as fluctuates naturally through the cell cycle. Here, we present a protocol to carry out long-term live-cell imaging of cells expressing a cytosolic Zn sensor. We then describe how to track cells using the published pipeline EllipTrack and how to analyze the single-cell traces to determine changes in labile Zn in response to perturbation.

Transient Zn deficiency induces replication stress and compromises daughter cell proliferation.

Cells must replicate their genome quickly and accurately, and they require metabolites and cofactors to do so. Ionic zinc (Zn) is an essential micronutrient that is required for hundreds of cellular processes, including DNA synthesis and adequate proliferation. Deficiency in this micronutrient impairs DNA synthesis and inhibits proliferation, but the mechanism is unknown.

Transient Zn deficiency induces replication stress and compromises daughter cell proliferation.

Cells must replicate their genome quickly and accurately, and they require metabolites and cofactors to do so. Ionic zinc (Zn) is an essential micronutrient that is required for hundreds of cellular processes, including DNA synthesis and adequate proliferation. Deficiency in this micronutrient impairs DNA synthesis and inhibits proliferation, but the mechanism is unknown.

Cellular zinc status alters chromatin accessibility and binding of transcription factor p53 to genomic sites.

Zinc (Zn) is an essential metal required by approximately 2500 proteins. Nearly half of these proteins act on DNA, including > 850 human transcription factors, polymerases, DNA damage response factors, and proteins involved in chromatin architecture. How these proteins acquire their essential Zn cofactor and whether they are sensitive to changes in the labile Zn pool in cells remain open questions.

Human cells experience a Zn pulse in early G1.

Zinc is an essential micronutrient required for all domains of life. Cells maintain zinc homeostasis using a network of transporters, buffers, and transcription factors. Zinc is required for mammalian cell proliferation, and zinc homeostasis is remodeled during the cell cycle, but whether labile zinc changes in naturally cycling cells has not been established.

Targeting Riboswitches with Beta-Axial-Substituted Cobalamins.

RNA-targeting small-molecule therapeutics is an emerging field hindered by an incomplete understanding of the basic principles governing RNA-ligand interactions. One way to advance our knowledge in this area is to study model systems where these interactions are better understood, such as riboswitches. Riboswitches bind a wide array of small molecules with high affinity and selectivity, providing a wealth of information on how RNA recognizes ligands through diverse structures.

Genetically encoded fluorescent sensors for metals in biology.

Metal ions intersect a wide range of biological processes. Some metal ions are essential and hence absolutely required for the growth and health of an organism, others are toxic and there is great interest in understanding mechanisms of toxicity. Genetically encoded fluorescent sensors are powerful tools that enable the visualization, quantification, and tracking of dynamics of metal ions in biological systems.

Single molecule microscopy to profile the effect of zinc status on transcription factor dynamics.

The regulation of transcription is a complex process that involves binding of transcription factors (TFs) to specific sequences, recruitment of cofactors and chromatin remodelers, assembly of the pre-initiation complex and recruitment of RNA polymerase II. Increasing evidence suggests that TFs are highly dynamic and interact only transiently with DNA. Single molecule microscopy techniques are powerful approaches for tracking individual TF molecules as they diffuse in the nucleus and interact with DNA.

Directed Evolution of a Bright Variant of mCherry: Suppression of Nonradiative Decay by Fluorescence Lifetime Selections.

The approximately linear scaling of fluorescence quantum yield (ϕ) with fluorescence lifetime (τ) in fluorescent proteins (FPs) has inspired engineering of brighter fluorophores based on screening for increased lifetimes. Several recently developed FPs such as mTurquoise2, mScarlet, and FusionRed-MQV which have become useful for live cell imaging are products of lifetime selection strategies. However, the underlying photophysical basis of the improved brightness has not been scrutinized.

Stage-specific differential expression of zinc transporter SLC30A and SLC39A family proteins during prostate tumorigenesis.

Prostate cancer (PCa) initiation and progression uniquely modify the prostate milieu to aid unrestrained cell proliferation. One salient modification is the loss of the ability of prostate epithelial cells to accumulate high concentrations of zinc; however, molecular alterations associated with loss of zinc accumulating capability in malignant prostate cells remain poorly understood. Herein, we assessed the stage-specific expression of zinc transporters (ZNTs) belonging to the ZNT (SLC30A) and Zrt- and Irt-like protein (ZIP) (SLC39A) solute-carrier family in the prostate tissues of different genetically engineered mouse models (GEMM) of PCa (TMPRSS2-ERG.

Characterization of Global Gene Expression, Regulation of Metal Ions, and Infection Outcomes in Immune-Competent 129S6 Mouse Macrophages.

Nutritional immunity involves cellular and physiological responses to invading pathogens, such as limiting iron, increasing exposure to bactericidal copper, and altering zinc to restrict the growth of pathogens. Here, we examine infection of bone marrow-derived macrophages from 129S6/SvEvTac mice by Salmonella enterica serovar Typhimurium. The 129S6/SvEvTac mice possess a functional Slc11a1 (Nramp-1), a phagosomal transporter of divalent cations that plays an important role in modulating metal availability to the pathogen.

Zn influx activates ERK and Akt signaling pathways.

Zinc (Zn) is an essential metal in biology, and its bioavailability is highly regulated. Many cell types exhibit fluctuations in Zn that appear to play an important role in cellular function. However, the detailed molecular mechanisms by which Zn dynamics influence cell physiology remain enigmatic.

Systematic Comparison of Vesicular Targeting Signals Leads to the Development of Genetically Encoded Vesicular Fluorescent Zn and pH Sensors.

Genetically encoded fluorescent sensors have been widely used to illuminate secretory vesicle dynamics and the vesicular lumen, including Zn and pH, in living cells. However, vesicular sensors have a tendency to mislocalize and are susceptible to the acidic intraluminal pH. In this study, we performed a systematic comparison of five different vesicular proteins to target the fluorescent protein mCherry and a Zn Förster resonance energy transfer (FRET) sensor to secretory vesicles.

Tools and techniques for illuminating the cell biology of zinc.

Zinc (Zn) is an essential micronutrient that is required for a wide variety of cellular processes. Tools and methods have been instrumental in revealing the myriad roles of Zn in cells. This review highlights recent developments fluorescent sensors to measure the labile Zn pool, chelators to manipulate Zn availability, and fluorescent tools and proteomics approaches for monitoring Zn-binding proteins in cells.

A multicolor riboswitch-based platform for imaging of RNA in live mammalian cells.

A key approach to investigating RNA species in live mammalian cells is the ability to label them with fluorescent tags and track their dynamics in the complex cellular environment. The growing appreciation for the diversity of RNAs in nature, especially the roles of small, non-coding RNAs for cell function, calls for development of orthogonal RNA tagging systems. We previously developed Riboglow, a new RNA tagging system that features modular elements and hence the possibility to customize features for each application of choice.

Single cell analysis reveals multiple requirements for zinc in the mammalian cell cycle.

Zinc is widely recognized as essential for growth and proliferation, yet the mechanisms of how zinc deficiency arrests these processes remain enigmatic. Here we induce subtle zinc perturbations and track asynchronously cycling cells throughout division using fluorescent reporters, high throughput microscopy, and quantitative analysis. Zinc deficiency induces quiescence and resupply stimulates synchronized cell-cycle reentry.

Dissociated Hippocampal Neurons Exhibit Distinct Zn Dynamics in a Stimulation-Method-Dependent Manner.

Ionic Zn has increasingly been recognized as an important neurotransmitter and signaling ion in glutamatergic neuron pathways. Intracellular Zn transiently increases as a result of neuronal excitation, and this Zn signal is essential for neuron plasticity, but the source and regulation of the signal is still unclear. In this study, we rigorously quantified Zn, Ca, and pH dynamics in dissociated mouse hippocampal neurons stimulated with bath application of high KCl or glutamate.

Remodeling of Zn homeostasis upon differentiation of mammary epithelial cells.

Zinc is the second most abundant transition metal in humans and an essential nutrient required for growth and development of newborns. During lactation, mammary epithelial cells differentiate into a secretory phenotype, uptake zinc from blood circulation, and export it into mother's milk. At the cellular level, many zinc-dependent cellular processes, such as transcription, metabolism of nutrients, and proliferation are involved in the differentiation of mammary epithelial cells.